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Title: Role of Promyelocytic Leukemia (PML) and Small Ubiquitin-like Modifier (SUMO) Proteins in Alternative Lengthening of Telomeres
Keywords: Promyelocytic leukemia, Alternative Lengthening of Telomeres
Issue Date: 25-Feb-2010
Citation: YONG WEI YAN, JACKLYN (2010-02-25). Role of Promyelocytic Leukemia (PML) and Small Ubiquitin-like Modifier (SUMO) Proteins in Alternative Lengthening of Telomeres. ScholarBank@NUS Repository.
Abstract: The Alternative Lengthening of Telomeres (ALT) mechanism of telomere maintenance is utilized in 10 to 15% of human tumors. ALT positive cancer cells have distinct morphological hallmarks such as heterogeneous telomere length, ALT-associated promyelocytic bodies (APBs) and extrachromosomal telomeric DNA. It is currently unknown how ALT is activated. The mechanisms through which ALT maintains and elongates telomere length are also unclear. As the Small Ubiquitin-like modifier (SUMO) protein modification of the promyelocytic leukemia (PML) protein is required for the formation of PML nuclear bodies, the SUMO pathway, particularly with regard to the formation of APBs, was investigated in an ALT context. While p53, an important tumor suppressor and a pro-apoptotic factor, is also modifiable by SUMO, such modification has not been investigated in an ALT context. Thus SUMOmodification of p53 in ALT cells was also investigated. Western blot analysis was used to show that p53 was ubiquitinated in a telomerase-positive human breast cancer cell line MCF7 while SUMOylated in an ALT cell line JFCF-6/T.1R. Results from the cell cycle and western blot analyses indicated that the tumor suppressive functions of p53, both endogenous and exogenous, were highly regulated by SUMOylation in human cancer cells. In addition, the use of arsenite as a source of oxidative stress inducer showed that under conditions of stress, the de-SUMOylation of p53 led to its transcriptional activation. Thus cellular conditions appeared to be critical in the SUMO regulation of the activities of p53. Through the use of a PML coiled coil domain deficient (PML C/C-) mutant which cannot be SUMOylated, our immunofluorescence data suggests that the coiled-coil domain is critical for the formation of APBs in ALT cells. The stable over-expression of PML led to a slight increase in telomere length while that of PML C/C- resulted in a moderate reduction in telomere length in ALT cells as observed from southern blot analysis. Wild-type PML and PML C/C- were also stably over-expressed in non-ALT MCF7 cells. We demonstrate for the first time that APBs were detected in MCF7 cells when PML was over-expressed stably. We also report the novel observation that the stable over-expression of wild-type PML and PML C/C- in MCF7 cells led to an ALT-like heterogeneous telomere phenotype. Our data suggests a possible switch in the telomere maintenance mechanism in MCF7 cells stably transfected with PML as the telomerase activity measured by the TRAP assay dropped drastically. This underscores a novel role for the PML protein in the activation of the ALT pathway in a telomerase-positive environment. The cell viability assays showed that U2OS cells stably transfected with PML C/C- were more susceptible to anti-cancer drugs while MCF7 cells that exhibited ALT-like phenotypes were more sensitive to doxorubicin. Thus our study implicates the mode of telomere maintenance mechanism in the susceptibility of cancer cells towards anti-cancer drugs. This study provided a better understanding on how SUMOylation in ALT cells can be altered, leading to p53 functional changes, according to cellular conditions. Importantly, this study contributed to the current understanding of how ALT can be activated in a telomerase positive background and how the telomere maintenance mechanism can affect susceptibility towards anticancer drugs.
Appears in Collections:Ph.D Theses (Open)

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